Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction.

Duchenne muscular dystrophy (DMD) is the most common form of the progressive muscular dystrophies characterized by defects of the dystrophin gene. Although primarily characterized by degeneration of the limb muscles, cardiomyopathy is a major cause of death. Therefore, the development of curative modalities such as gene therapy is imperative. We evaluated the cardiomyopathic features of mdx mice to observe improvements in response to intravenous administration of recombinant adeno-associated virus (AAV) type 9 encoding microdystrophin. The myocardium was extensively transduced with microdystrophin to significantly prevent the development of fibrosis, and expression persisted for the duration of the study. Intraventricular conduction patterns, such as the QRS complex duration and S/R ratio in electrocardiography, were also corrected, indicating that the transduced microdystrophin has a protective effect on the dystrophin-deficient myocardium. Furthermore, BNP and ANP levels were reduced to normal, suggesting the absence of cardiac dysfunction. In aged mice, prevention of ectopic beats as well as echocardiographic amelioration was also demonstrated with improved exercise performance. These findings indicate that AAV-mediated cardiac transduction with microdystrophin might be a promising therapeutic strategy for the treatment of dystrophin-deficient cardiomyopathy.

Yeast-immobilized SPV device for koji quality control in sake brewing process.

The malted rice, koji, is an indispensable material for the brewing of sake. It saccharifies rice starch and supplies vitamins for the yeast in sake brewing. Since the quality of sake depends strongly on the quality of koji, quality control of koji is very important in the brewing. There are some methods to measure the activity of enzymes and the quantity of vitamins with the quality of koji. None of these methods, however, directly relate to the yeast metabolism. We constructed a sensor system to monitor the yeast metabolism in sake brewing by use of immobilized Saccharomyces cerevisiae and a Surface PhotoVoltage device (SPV). In this system, S. cerevisiae K701 and K9, designed for use in sake brewing by the Brewing Society of Japan, were employed as immobilized microbe. The pH change due to the production of organic acids in sake brewing is measured using the SPV. A linear relationship was observed between decrease in the photocurrent (the metabolism response) and the concentration to less than 60 mM of glucose (r=0.990). Then we measured the koji extract and observed the difference of response between K701 and K9 which corresponded to the productivity of acidic substances by batch test.

Expression of asparagine synthetase mRNA through asparagine independent signal transduction pathway that might involve protein kinase C in BALB3T3 cells.

Basal level of asparagine synthetase mRNA in BALB3T3 cells was elevated when the cells were shifted from medium containing a high concentration (3.3 mM) of asparagine to one lacking asparagine. We then studied whether the expression of asparagine synthetase mRNA is also mediated through other asparagine-independent signaling pathways. BALB3T3 cells grown to near confluence were quiesced by serum-starvation, and various agents were then added to the culture to examine the enzyme activity and mRNA level of asparagine synthetase. 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of protein kinase C (PKC), elevated dose and time dependently the level of asparagine synthetase mRNA even in Eagle's minimum essential medium with alpha modification (MEM alpha) that contains protein-constituting 20 amino acids and is supplemented with 3.3 mM asparagine. Staurosporine and H-7, PKC inhibitors, strongly blocked the fetal bovine serum-dependent accumulation of asparagine synthetase mRNA. TPA could also enhance the activity of asparagine synthetase within 24 h at concentrations of more than 10 nM. These results suggest that expression of asparagine synthetase gene can be induced both through a pathway that involves PKC and through a pathway the origin of which is a reduced concentration of asparagine in BALB3T3 cells.

Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice.

BACKGROUND: While tumour necrosis factor alpha (TNF-alpha) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. METHODS: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. RESULTS: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-alpha administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. CONCLUSIONS: Enhancement of the TNF-alpha/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Induction of asparagine synthetase by follicle-stimulating hormone in primary cultures of rat Sertoli cells.

We studied effects of various agents, including follicle-stimulating hormone (FSH), on changes in the level of asparagine synthetase in cultured rat Sertoli cells. FSH, dibutylyl cAMP (Bt2cAMP), and cAMP-increasing agents, such as forskolin and theophylline, increased enzyme activity within 24 h. Western blot analysis revealed the increase in cellular protein content of asparagine synthetase by these agents to a degree similar to that of the enhanced enzyme activity. Northern blot analysis also showed elevation of the level of mRNA for asparagine synthetase in FSH- and Bt2cAMP-treated cells. Testosterone, insulin, prostaglandin E2, or 12-O-tetradecanoyl phorbol-13-acetate did not stimulate the enzyme. The results suggest that asparagine synthetase is induced via a cAMP-dependent signal transduction pathway in Sertoli cells. Increased production of asparagine in FSH- and Bt2cAMP-treated cells was indicated by extracellular accumulation of more asparagine around the treated cells than around the controls. The basal level of asparagine synthetase assessed in extracts from whole testes increased with age of animals toward 60-70 days, but the degree of hormonal stimulation of the enzyme in isolated Sertoli cells decreased during Days 20 to 30 postpartum. This suggested some different mechanism for regulation of the enzyme. The enhanced activity of asparagine synthetase by FSH in Sertoli cell may be important for maturation and function of Sertoli cells.

Cloning of cDNA for asparagine synthetase from rat Sertoli cell.

We have cloned and sequenced cDNA of asparagine synthetase (AS) from rat Sertoli cells. The nucleotide sequence was derived by analysis of cloned cDNA of reverse transcription-polymerase chain reaction (RT-PCR) product that spanned overall the cDNA coding region. The sequence contains three nucleotide differences when compared with that of rat Fao hepatoma cells (Hutson, R.G., and Kilberg, M.S. (1994) Biochem. J. 304, 745-750). Accordingly, amino acid residues Ser at position 330 and His at 491 of Sertoli cells were replaced by Pro and Tyr, respectively, in the sequence of the hepatoma cells. Mutational nucleotide changes may occur during carcinogenesis. The testis contained the most abundant AS mRNA among the tissues studied and others revealed by far a little amount of message. Expressions of AS mRNA in the liver and brain were high in fetal period and reduced rapidly after birth, showing importance of AS in cell proliferation.